Our aim is to understand the molecular mechanisms by which Drosophila counts its X chromosomes to determine sex. The research addresses three problems of broad significance: The function of dose-sensitive transcriptional switches, the means by which different classes of regulatory factors function together to control gene expression, and the molecular basis of sexual development. The proposed research focuses on the early expression of Sex-lethal, the master regulatory gene governing sex determination and dosage compensation. The emphasis is on the switch-like response of the establishment promoter, SxlPe, to X chromosome dose. SxlPe is turned on in XX embryos (females) and left off in XY males. Although several positive and negative regulators are involved in assessing the two-fold difference in X dose that sets on-or-off response of SxlPe, the X-linked, sisB and sisA genes, are the key quantitative determinants. The sisB gene, known in its proneural guise as scute; encodes a bHLH protein that recognizes canonical E-box and non-canonical DNA binding sites at SxlPe. The role of each important site will be tested in vivo using P-element transfonnation to determine if they function as dose-sensing or transcription augmentation elements. For those that function as dose-sensors we will determine whether DNA binding affinity or other factors are the crucial feature controlling the dose-response. The sisA gene encodes a unique bZIP protein. Nothing is known of how sisA protein interacts with SxlPe. We will isolate the protein dimer partner of SISA and study its DNA binding at SxlPe. The molecular functions of these sites and the role of binding site affinity in dose sensing will be tested using transgenic SxlPe reporter constructs. Despite the many differences between insect and mammalian sex determination, both systems are exquisitely sensitive to the dose of several key regulators suggesting there may be hidden commonalities in the regulatory logic underlying them. In humans, these dose-sensitive interactions manifest themselves as diseases of sex-reversal and sterility. Because these, and many other diseases, occur due to two-fold reductions in gene dose, as opposed to the complete loss of gene function, it is important to understand how two-fold differences in gene dose can be assessed, and how the responses to such differences can be experimentally manipulated. In its elemental form, Drosophila sex determination serves as a text book example of how dose-sensitive interactions control developmental fates. Detailed understanding of the mechanics and circuitry underlying the process will prove even more instructive.